We performed studies to examine the role of nitric oxide in cardioprotection. Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC). Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion;(2) IPC;or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca2+ channel alpha1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca2+-ATPase, alpha-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase alpha1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart. It has been proposed that SNO may provide cardioprotection, in part, by reducing cysteine oxidation during ischemia-reperfusion (IR) injury. To test this hypothesis, we developed a new method to measure oxidation using resin assisted capture (RAC), similar to the SNO-RAC methods used in the quantification of S-nitrosylation. Langendorff perfused hearts were subjected to various perfusion protocols (control, IPC, IR, IPC-IR) and homogenized. Each sample was divided into two equal aliquots, and a modified biotin switch was performed in order to simultaneously analyze SNO and oxidation. Using two independent measures, we identified 44 different proteins which showed increased S-nitrosylation with IPC. The majority (40) of these proteins also showed a decrease in cysteine oxidation following IR. We identified 53 cysteine residues from these 40 proteins that showed a decrease in oxidation at the same site of S-nitrosylation. These proteins included glyceraldehyde-3-phosphate dehydrogenase, electron transfer flavoprotein , and voltage-dependent anion-selective channel protein 2. Further, in an in vitro assay, oxidative challenge of purified alpha-ketoglutarate dehydrogenase increased the production of reactive oxygen species (ROS) by more than 60%. Interestingly, pretreatment with the S-nitrosylating agent S-nitrosoglutathione prevented the oxidation-induced increase in ROS production. These results suggest that SNO yields cardioprotective effects through two distinct mechanisms: (1) by directly blocking against the ROS-induced oxidation of cysteine residues, and (2) by reducing the production of ROS.